Crime Scene Investigator PCR Basics™

Stan HitomiCoordinator – Math & ScienceSan Ramon Valley Unified School DistrictDanville, CA

Kirk BrownLead Instructor, Edward Teller Education CenterScience Chair, Tracy High School and Delta College, Tracy, CA

Sherri Andrews, Ph.D.Curriculum and Training SpecialistBio-Rad Laboratories

Essy Levy, M.Sc.Curriculum and Training SpecialistBio-Rad Laboratories

Crime Scene Investigator PCR Basics™ Kit

Instructors

Why Teach Crime Scene Investigator PCR Basics™ Kit ?

•Exciting real-world connections

•Tangible results

•Statistical Analysis

•Standards-based

Target Audience

•The Crime Scene Investigator PCR Basics™ Kit is intended to be an introduction to the polymerase chain reaction (PCR)

•Students will have a much better appreciation of the kit if they have some understanding of DNA structure and function

Crime Scene Investigator PCR Basics™Kit Advantages

•Standards Based

•Perform real-world DNA profiling •Use PCR to amplify multiple DNA samples

•Use electrophoresis to visualize results

•Complete in two 45 minute lab sessions •Sufficient materials for 8 student workstations

Workshop Time Line • Introduction to DNA profiling

• Set up PCR reactions

• Electrophorese PCR products

• Analysis and interpretation of results

What is DNA profiling?

DNA profiling is the use of molecular genetic methods to determine the exact genotype of a DNA sample in a way that can basically distinguish one human being from another

The unique genotype of each sample is called a DNA profile.

How do crime scene investigators create a DNA profile?

1. Evidence is collected at the crime scene:

Blood Tissue Semen

Urine HairTeeth Saliva Bone

2. DNA is extracted from sources at the crime scene and from victim and suspects

How do crime scene investigators create a DNA profile?

How do crime scene investigators create a DNA profile?

3. DNA samples are processed

Sample Obtained from Crime Scene or

Paternity Investigation Biology

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof multiple (STR) markers

PCR Amplificationof multiple (STR) markers

Technology

Separation and Detection of PCR Products

(STR Alleles)

Separation and Detection of PCR Products

(STR Alleles)

Sample Genotype Determination

Sample Genotype Determination

Genetics

Comparison of Sample Genotype to Other Sample

Results

Comparison of Sample Genotype to Other Sample

Results

If match occurs, comparison of DNA profile to population

databases

If match occurs, comparison of DNA profile to population

databases

Generation of Case Report with Probability of Random

Match

Generation of Case Report with Probability of Random

Match

Since humans are 99.9% identical where do crime scene investigators look for differences in DNA profiles?

4. Crime Scene Investigators search in areas of the genome that are unique from individual to individual and are “anonymous” (control no known trait or function) The areas examined are Short Tandem Repeats or STR’s

STR region

Example of an STR: TH01

The TH01 locus contains repeats of TCAT.

CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA

This example has 6 TCAT repeats.

There are more than 20 known TH01 alleles.

Each individual inherits 1 allele from each parent.

Determining genotypes for individuals using STRs

Ms. Smith’s TH01 locus for her two chromosomes is given below.

What is her genotype?

MOM’S CHROMOSOME

CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA

DAD’S CHROMOSOME

CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT AAA

To determine the genotype (DNA profile) Crime Scene Investigators make billions of copies of the target sequence using PCR

Starting DNA Template

5’

5’

3’

3’

Target DNA

What’s the point of PCR?

•PCR, or the polymerase chain reaction, makes copies of a specific piece of DNA

•PCR allows you to look at one specific piece of DNA by making copies of *only* that piece of DNA

•PCR is like looking for a needle in a haystack, and then making a haystack out of the needle

DNA profiling is used to determine which suspect can not be excluded from suspicion.

How are suspects included or excluded from an investigation?

•Suspects are included in an investigation if their DNA profile matches with genotypes found at the crime scene

•Suspects can be excluded if their DNA profile does not match genotypes found at the crime scene

Crime Scene Investigator PCR Basics™ ProceduresOverview

LaboratoryQuick Guide

Set up PCR reactions

1. Find the PCR tubes at your station. Label them ‘CS’ for Crime Scene DNA, ‘A’ for Suspect A DNA, ‘B’ for Suspect B DNA, ‘C’ for Suspect C DNA, and ‘D’ for Suspect D DNA.

2. Keeping the tubes on ice, add 20 μl of Master Mix + blue primers to each tube.

3. Keeping the tubes on ice, add 20 µl of each DNA to the appropriately labeled tube.

4. USE A FRESH TIP EACH TIME!

5. Mix and put in thermal cycler

6. Cycle ~3 hours

The PCR Reaction

What do you need?

What is needed for PCR?

• Template (containing the STR you want to amplify for the study)

• Sequence-specific primers flanking the target sequence

• Nucleotides (dATP, dCTP, dGTP, dTTP)

• Magnesium chloride (enzyme cofactor)

• Buffer, containing salt• Taq polymerase

5’

3’5’

3’3’ 5’

3’5’

Forward primer

Reverse primer

Target sequence

What is happening in the PCR tube while in the thermocycler? PCR Animation

http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html

The PCR Reaction

How does it work?

Heat (94oC) to denature DNA strands

Cool (52oC) to anneal primers to template

Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA

Repeat 35 cycles

Denaturing Template DNA

Heat causes DNA strands to separate

3’

5’

5’

3’

Denaturation of DNA at 94oC

5’

3’

3’

5’

Annealing Primers

Primers bind to the template sequence

Taq polymerase recognizes 3’ end of primer + template strand

Primers anneal at 52oC

5’

3’

3’

5’

5’

3’

3’

5’

3’5’3’ 5’

3’ 5’3’5’

Taq extends at 72oC

STR DNA is replicated

Repeat denaturing, annealing, and extending 35 cycles

Cycle 1

Cycle 2

Cycle 3The exact-length target product is made in the third cycle

To visualize PCR products Crime Scene investigators use gel electrophoresis

(14)

(12)

(11)

(9)

(8)

(7)

(6)

(5)

(4)

(3)

(13)

(10)

TH01 alleles

Alleleladder

Mother Father Child C Child D Child E

Electrophorese PCR products

1. Add 10 ul of Orange G Loading Dye to each PCR tube and mix

2. Set up gel and electrophoresis equipment

3. Load 20 ul of CSI allele ladder to Lane 1

4. Load 20 ul of your PCR reactions in lanes 2 to 6

5. Electrophorese samples

6. Stain gel with Fast Blast DNA Stain

7. Analyze results

Using the digital micropipet

Add 10ul of loading dye to each microtube

AgaroseElectrophoresis

Place gel in gel box

Pour buffer in box until gel wells are covered.

Place 20ul of samples into appropriate wells

Set up electrophoresis chamber by putting top in place and connecting it to the power supply

Gel running

AgaroseElectrophoresisRunning

Agarose gel sieves DNA fragments according to size– Small fragments

move farther than large fragments

Use a 3% gel to

separate small fragment sizes

Milestones in Forensic DNA analysis

1985 Alec Jeffries develops RFLP

1990 PCR analysis using single locus STR begins

1992 FBI initiates STR work

1994 DNA Identification Act: provides funding for national DNA database

1995 OJ Simpson trial focuses public attention on DNA evidence

1998 FBI starts CODIS database; Swissair disaster – all remains identified using STR DNA profiling

2001 World Trade Center disaster in NYC – many remains identified using a combination of DNA profiling approaches

2004 Indian Ocean tsunami; Interpol and other world agencies use DNA profiling to identify

victims

Today Trace your Genetic Genealogy; commercially available packages can trace paternal/maternal ancestry

DNA Testing Today

GeneTree.com & Ancestry.com provide DNA tests from $99-$200 to trace genealogy

Genealogical Analysis Uses:DNA sequencing (mtDNA)

Single Nucleotide Polymorphism testing (SNPs)

Short Tandem Repeat testing (STRs)

Crime scene investigators use techniques that are fast, cost effective, and have a high Power of Discrimination

The Power of Discrimination is the ability of a test to distinguish between different samples (genotypes)

slow fast

low

high

mtDNAmtDNA Blood group Blood group typingtyping

RFLP RFLP analysisanalysis

STR STR analysisanalysis

Speed of Analysis

Pow

er

of

Dis

crim

inati

on

Statistics of Chance: M&M Locus

6 Possible Alleles:•Green•Red•Yellow•Blue•Brown•Orange

Probabilities• One allele from each parent means 2

copies of gene/locus

1

6

1

6X =

1

36

Frequency of any M&M genotype

Probabilities

Mike & Ikes: 5 alleles

Jolly Rancher: 5 alleles

1

6

1

6

1

5

1

5

1

5

1

5

1

36

1

25

1

25

Locus

M&M6 alleles

Jolly Rancher5 alleles

Mike & Ikes5 alleles

x =

x =

x =

1

22,500=Chance an individual has a given genotype

1

506,250,000=Chance 2 people have

the same genotype

Who can’t we exclude from the pool of suspects?

The Power of Discrimination increases with the number of loci profiled

TPOX genotype8-12 D3S1358 genotype 16-17

FGA genotype 21-23

0.099

0.050

0.044

0.044 x 0.099 x 0.050 = 2.18 x 10-4

1 / 2.18 x 10-4

or 1 in 4591

The Power of Discrimination increases with the number of loci profiled

TPOX 8-12 0.044

D3S1358 16-17 0.099

FGA 21-23 0.050

VWA 14-14 0.0088

Random Match Probability

= 1 in 5.3 x 105

Real-WorldProbabilities •Forensics labs use 13 different loci

with multiple alleles

•Allele frequencies DO NOT follow mathematical principles - allele frequencies vary by population.

•These 13 loci allow for discrimination of any two people in the world (with the exception of identical twins), living or dead.

•Probability of a random match when all 13 loci typed: ~1 in 3 trillion.

Butler et al 2003 J Forensic Sci. www.cstl.nist.gov/biotech/strbase/pub_pres/Butler2003a.pdf

Allele Caucasians

n=302

African American

n=258

Latinos

n=1405 .002 .004

6 .232 .124 .214

7 .190 .421 .279

8 .084 .194 .096

9 .114 .151 .150

10 .008 .002 .014

11 .002

TH01 Published Allele Frequencies by Population

CODIS COmbined DNA Index System

A federally maintained database used by law enforcement officials

13 loci guarantees high power of discrimination

Real STR analysis

Four different fluorescent tags have been used to identify 7 amplified loci

Allele ladders are indicated by arrows

Analysis of Results:

Who can’t be excluded?

107

5

4

3

2

1

CS A B C D

genotype

5-2 7-4 5-2 7-2 10-3

AL: Allele ladderCS: Crime SceneA: Suspect AB: Suspect BC: Suspect CD: Suspect D

AL

15

BX

P00

7 a

llel

es